CN108102972A - A kind of preparation method and application of the compound biocontrol fungicide of cure plant disease of peanut - Google Patents
A kind of preparation method and application of the compound biocontrol fungicide of cure plant disease of peanut Download PDFInfo
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Abstract
Invention provides a kind of preparation method of the compound biocontrol fungicide of cure plant disease of peanut, and strain component is Brevibacillus laterosporus LX12, and deposit number is CGMCC 7420 and bacillus subtilis LX13, and deposit number is CGMCC No.8805；Its preparation method cultivates 3d will to be inoculated into 28 DEG C of LB solid mediums after above-mentioned actication of culture respectively；Beef extract-peptone fluid nutrient medium is taken, is respectively connected to the Brevibacillus laterosporus LX12 fermentation seed liquids of above-mentioned preparation and bacillus subtilis LX13 fermentation seed liquids, in 30~40 DEG C of pH7.2~8.5, temperature, 1~2d is to get production bacterium solution for fermentation；According to the volume ratio of Brevibacillus laterosporus LX12 bacterium solutions and bacillus subtilis LX13 bacterium solutions it is 2 by bacterium solution:1 proportioning mixing, is made compound biocontrol fungicide.Compound biocontrol fungicide provided by the invention has apparent control effect to crown rot, southern blight and root rot；Solid compound biocontrol fungicide solves the problems, such as the recycling of peanut vine, additionally it is possible to live away from home space and nutriment is provided for biocontrol microorganisms, moreover it is possible to improve soil property.
Description
Technical field
The invention belongs to microbial technology fields, and in particular to a kind of preparation of the compound biocontrol fungicide of cure plant disease of peanut
Method and application.
Background technology
Peanut crown rot is a kind of peanut time of infertility generable soil-borne fungus type disease, and cause of disease is
Aspergillus niger V.Tiegh belong to Deuteromycotina, hyphomycetales Eurotium, black-koji mould fungi.The disease is mainly sent out
It gives birth in seedling stage or grows early period.In peanut seedling stage, germ first infects the cotyledon of remaining, and then the basal part of stem of infecting peanut.Sick portion
The concave scab of nascent yellowish-brown, scab edge brown, later disease portion expand rapidly, and cortex lobe organizes dry rot, finally only surplus
Broken fibr tissue down.In the case of humidity, sick portion grows the mould layer of black quickly, i.e., the mitogenetic spore of germ is in stalk and mitogenetic
Spore.Kernel is infected, can be allowed to rot and cannot germinate, one layer of black mould is grown in aggrieved portion faces.After benevolence germination, disease
Bacterium can infect cotyledon, and cotyledon is not unearthed and rots with regard to blackening, and it is in water soaking mode that plumular axis, which is infected, light brown, there is the mould layer of black.Peanut
Growth period is susceptible, often results in basal part of stem and rots, and diseased plant is wilted withered.
Peanut sclerotium rolfsii is the important disease on peanut, and cause of disease is Sclerotium rolfsii Sacc., belongs to Fungi Imperfecti
Subphylum, no spore Zoopagales, Sclerotium rolfsii category fungi.The disease mostly occurs in Adult plant, and hot and humid 7~September part is that morbidity is contained
Phase, main harm basal part of stem, carpopodium, fruit pod and root.Aggrieved diseased tissues initial stage is in the soft corruption of crineous, grows white spun silk soon
The mycelia of shape is covered by sick position.Environmental condition suitable for when, mycelia outwards spreads rapidly, the cane of peanut middle and lower part near the ground
And the soil surface around diseased plant, it can all grow one layer of bombycine subiculum of white；Later stage is formed in sick portion's subiculum
Many sclerotium；With rotting for aggrieved diseased tissues, plant cortex comes off, only remaining fibr tissue, very frangibility.After pod is susceptible
Sick portion becomes light brown to crineous, becomes shrinkage after kernel is susceptible and rots, sick portion covers taupe subiculum, and the later stage can also form bacterium
Core；Germ inside kind of shell and benevolence surface growth when can also generate oxalic acid so that on kind of skin formed striped, sheet or
Circular black-and-blue lathe work.
Peanut root rot can occur in peanut each breeding time, and cause of disease is Deuteromycotina Fusarium fungi.Before emergence
Rotten kind, rotten bud can be caused by catching an illness；It is aggrieved in seedling stage to cause seedling withered；Cause root-rot, brown foot rot and pod rotten Adult plant is aggrieved,
The short and small, undergrowth of diseased plant overground part performance, blade from bottom to top dry up after gradual change Huang, main root browning, shrinkage, dry rot, lateral root
It comes off or lateral root is few and short, main root only stays the root tissue of remaining, be commonly called as its " mouse tail " as mouse tail when pulling up.
Currently to the prevention of peanut pest and disease damage mainly based on plant resistance to environment stress kind and chemical prevention, but Pest-resistant
Problem getting worse, and chemical pesticide residual causes serious harm, and such as residual hazard, environmental pollution, human body and ecology are caused
Very big threat, is unfavorable for the sustainable development of peanut cultivation industry.Therefore, a kind of economic, safe and effective control measure is found
It is extremely urgent.Bio-control method is to be killed or reduced the quantity of causal organism using beneficial microbe to control plant disease
Occur, a kind of measure of development.These beneficial organisms are also known as antagonistic microbe or biocontrol microorganisms.It is related to peanut biocontrol microorganisms at present
Research, still, for above-mentioned three cultivate peanut pathogen biocontrol agent in application process there are preventive effect it is unstable, work it is slow,
The problem of long action time, single preventive and therapeutic effect so that the application of microbial bacterial agent receives obstruction.
The content of the invention
In view of the above-mentioned problems, the present invention is intended to provide a kind of preparation method of compound biocontrol fungicide, acts on peanut rhizosphere,
Effect with extraordinary prevention peanut crown rot, southern blight and root rot.
The present invention is achieved by the following technical solutions：
A kind of preparation method of the compound biocontrol fungicide of cure plant disease of peanut, strain component for Brevibacillus laterosporus-
LX12, deposit number are CGMCC 7420 and bacillus subtilis-LX13, and deposit number is CGMCC No.8805；It is made
Preparation Method cultivates 3d will to be inoculated into 28 DEG C of LB solid mediums after above-mentioned actication of culture respectively；Beef extract-peptone liquid is taken to train
Base is supported, is respectively connected to the Brevibacillus laterosporus-LX12 fermentation seed liquids of above-mentioned preparation and bacillus subtilis-LX13 fermentations kind
Sub- liquid, in 30~40 DEG C of pH7.2~8.5, temperature, 1~2d is to get production bacterium solution for fermentation；It prepared by above-mentioned steps various
Production bacterium solution is 2 according to the volume ratio of Brevibacillus laterosporus-LX12 bacterium solutions and bacillus subtilis-LX13 bacterium solutions:1
Proportioning mixing, is made compound biocontrol fungicide.
Further, charcoal is prepared by raw material of peanut vine, charcoal crushed 30 mesh sieves obtains biological powdered carbon, will
It is 1 that biological powdered carbon, which is obtained, according to solid-to-liquid ratio:The ratio of (1~3) is put into compound biocontrol fungicide, is stirred evenly, air drying,
Obtain solid compound biocontrol fungicide.
Further, charcoal is prepared by raw material of peanut vine, charcoal crushed 30 mesh sieves obtains biological powdered carbon, will
It is 1 that biological powdered carbon, which is obtained, according to solid-to-liquid ratio:The ratio of (1~3) puts into Brevibacillus laterosporus-LX12 bacterium solutions and withered grass respectively
In bacillus-LX13 bacterium solutions, the volume of the Brevibacillus laterosporus-LX12 bacterium solutions and bacillus subtilis-LX13 bacterium solutions
Than for 2:1, it stirs evenly, air drying, obtains solid biological and ecological methods to prevent plant disease, pests, and erosion single dose, two kinds of single doses are mixed to get solid compound biological and ecological methods to prevent plant disease, pests, and erosion
Microbial inoculum.
The present invention also provides application of the above-mentioned compound biocontrol fungicide in terms of prevention peanut crown rot, southern blight and root rot
And the application in terms of peanut growth and output increased is promoted.
Further, respectively the sowing time in peanut, after planting 10d using pouring root after compound biocontrol bacterium dilution agent or
The sowing time of peanut carries out the sowing of solid union biocontrol agent.
Further, by 100 times of compound biocontrol bacterium dilution agent, 100ml is poured every time per pier peanut.
Compared with prior art, compound biocontrol fungicide provided by the invention has apparent antagonism to peanut pathogen,
Potentiality biocontrol bacterial strain can be used as；Biocontrol effect is good, has apparent control effect to crown rot, southern blight and root rot；It can
It is obviously promoted growth and the yield of peanut；It is easy to use, it is rapid-action, convenient for applying and promoting；Solid compound biocontrol fungicide,
The recycling problem of peanut vine is not only solved, but also live away from home space and nutriment can be provided for biocontrol microorganisms, extends and makees
With the time, convenient for preserving, moreover it is possible to improve soil property, improve peanut yield and quality.
Description of the drawings
Fig. 1 is the colonial morphology figure of bacterial strain LX13；
Fig. 2 is the PCR product amplification figure of the 16s rDNA of bacterial strain LX13；
Fig. 3 be bacterial strain LX13 with its in GenBank databases it is related belong to plant build based on 16S rDNA sequences
Phylogenetic tree.
Biological material specimens preservation information：
LX12 strains in the present invention, have disclosed in the patent of Application No. 201310643869.3.The strain is
It survived after testing on 04 07th, 2013, deposit number is CGMCC 7420.Classification And Nomenclature is Brevibacillus laterosporus
(Brevibacillus laterosporus), entitled China Committee for Culture Collection of Microorganisms of depositary institution is commonly micro-
Bio-Centers (CGMCC), address be city of BeiJing, China Chaoyang District North Star West Road 1 institute 3, Chinese Academy of Sciences's microbe research
Institute.
LX13 strains in the present invention, preservation date are on 01 22nd, 2014, deposit number CGMCC No.8805.Point
Class is named as bacillus subtilis (Bacillus subtilis), the entitled Chinese microorganism strain preservation management of depositary institution
Committee's common micro-organisms center (CGMCC), address are the institute 3 of BeiJing, China Chaoyang District North Star West Road 1, and the Chinese Academy of Sciences is micro-
Biological study institute.
Specific embodiment
The present invention is described in further details with reference to specific embodiment and attached drawing.
Material source and preparation method are as follows used by following embodiment：
1st, microbial strains and its source
Bacterial strain：Brevibacillus laterosporus-LX12 (CGMCC No.7420)；Bacillus subtilis-LX13 (CGMCC
NO.8805)；2 kinds of microbial strains are separated by Shandong Peanut Inst., have been preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center.
2nd, disease fungus source is tested
Shandong Peanut Inst. preserves peanut crown rot bacterium, sclerotium rolfsii, pine root fungus
3rd, culture medium
(1) isolation medium：NA culture mediums：Beef extract (Beef extract) 3g, peptone (Peptone) 5g, Portugal
Grape sugar (Dextrose) 10g, agar powder (Agar) 15g, distilled water 1000mL adjust pH to 7.0,121 DEG C of high pressure steam sterilizations
20min。
(2) activation medium
LB solid mediums：Tryptone (Tryptone) 10g, yeast extract (Yeast extract) 5g, sodium chloride
(NaCl) 10g, agar powder 15g, distilled water 1000mL adjust pH to 7.0,121 DEG C of high pressure steam sterilization 20min
PDA solid mediums：Potato 200g, glucose 20g, agar powder 15g, distilled water 1000mL, 121 DEG C of high pressures are steamed
Vapour sterilizing 20min.
(3) seed culture medium：
LB fluid nutrient mediums：Tryptone (Tryptone) 10g, yeast extract (Yeast extract) 5g, sodium chloride
(NaCl) 10g, distilled water 1000mL adjust pH to 7.0,121 DEG C of high pressure steam sterilization 20min.
PDA liquid medium：Potato 200g, glucose 20g, distilled water 1000mL, 121 DEG C of high pressure steam sterilizations
20min。
(4) disease fungus culture application PDA culture medium：Potato 200g, glucose 20g, agar powder 15g, distilled water
1000mL, 121 DEG C of high pressure steam sterilization 20min.
Separation, culture and the identification of embodiment 1LX13 bacterial strains
1st, the screening and separation of LX13 bacterial strains
A collection of single bacterium on picking tablet falls within setting-out on LB solid medium tablets and is inverted in 28 DEG C of constant incubator trainings
It supports 1-2 days.
Soil comes from Shandong Peanut Inst.'s experimental field peanut rhizosphere soil.Weighing 5g rhizosphere soils, to be put into 45ml sterile
In the triangular flask of water, a moment is shaken, upper liquid 0.5ml is drawn and is added to containing in 4.5ml sterile water test tubes, according to 102、103、
104、105、106Multiple carry out gradient dilution.
The 100 μ l of soil supension of different extension rates are drawn respectively with micropipettor in the spreader that on NA tablets, sterilizes
Coating hooks, and is inverted in after tablet is sealed in 28 DEG C of constant incubators and cultivates 48h.
A collection of single bacterium on picking tablet falls within setting-out on LB solid medium tablets and is inverted in 28 DEG C of constant incubator trainings
Support 1-2d.
2nd, the Physiology and biochemistry of the morphologic observation of bacterium colony and bacterial strain is identified
Biocontrol bacteria bacterial strain LX13 is grown on LB tablets, milky, and dry tack free has gauffer, matt, rough, bacterium
Fall edge to be irregularly serrated, as shown in Figure 1；Bacterial strain LX13 is in clear LB fluid nutrient mediums, and shaken cultivation, surface is not
Film layer is formed, bacteria suspension is muddy.
The ginsengs such as Gram's staining, V-P measure, catalase test, nitrate reduction, citrate utilization, methyl red test
According to《Common bacteria system identification handbook》Carry out Physiology and biochemistry identification.
The Physiology and biochemistry qualification result of LX13 bacterial strains is shown in Table 1, and the results show LX13 thalline show as Gram-positive, aerobic
Bacterium；Methyl red, catalase and V-P reactions are positive；Orthonitric acid can be gone back using citrate as the source of nutrition of carbon
Salt, caseinhydrolysate, starch and gelatin；And sugar alcohol can be fermented and generate acid, but edwardsiella hoshinae.According to more than morphological feature and
Physio-biochemical characteristics, and combine primary Jie Shi Bacteria Identifications handbook and common bacteria system identification handbook, biocontrol bacteria bacterial strain LX13
It is sufficiently close to bacillus subtilis (Bacillus subtilis).
The physiological and biochemical property of 1 active bacterial strain LX13 of table
Note："+" represents reaction result for the positive；"-" represents reaction result for feminine gender.
3rd, the 16S rRNA molecules identification of bacterium
Bacterial strain DNA is extracted
The bacterium bacterial strain that there is antagonistic activity to fungi is chosen, 10 identical bacterium colonies of picking do repetition, carry out genome
The extraction of DNA, extracting method are carried out referring especially to TIANGEN TIANamp BACTERia DNAKit kit specifications,
And change slightly, step is as follows：
(1) inoculum 1mL, 10000rpm centrifugation 1min is taken, exhaust supernatant as far as possible；
(2) 200 μ L buffer solution GA are added in into bacterial sediment, shakes to thalline and thoroughly suspends；
(3) 4 μ LRNAase (100mg/mL) solution are added in, 15s is shaken, is placed at room temperature for 5min；
(4) 20 μ L Proteinase K Solutions, mixing are added in into pipe；
(5) 220 μ L buffer solution GB are added in, shake 15s, 70 DEG C of placement 10min, brief centrifugation is to remove the water of cap wall
Pearl；
(6) 220 μ L absolute ethyl alcohols are added in, fully shaking mixing 15s is likely to occur flocculent deposit at this time, brief centrifugation with
Remove the droplet of cap wall；
(7) solution obtained by previous step and flocculent deposit all add in an adsorption column GB3 to (adsorption column is put into collecting pipe
In), 12000rpm centrifugation 30s, outwelling waste liquid will be put into adsorption column CB3 in collecting pipe；
(8) 500 μ L buffer solutions GD (whether preoperation inspection is added into absolute ethyl alcohol) are put into adsorption column CB3,
12000rpm centrifuges 30s, and outwelling waste liquid will be put into adsorption column CB3 in collecting pipe；
(9) 700 μ L rinsing liquids PW (whether preoperation inspection is added into absolute ethyl alcohol) are put into adsorption column CB3,
12000rpm centrifuges 30s, and outwelling waste liquid will be put into adsorption column CB3 in collecting pipe；
(10) 500 μ L rinsing liquids PW, 12000rpm centrifugation 30s are put into adsorption column CB3, outwell waste liquid by adsorption column
It is put into CB3 in collecting pipe；
(11) adsorption column CB3 is put back in collecting pipe, 12000rpm centrifugation 2min outwell waste liquid, adsorption column CB3 is placed in
It is placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in sorbing material；
(12) adsorption column CB3 is transferred in clean centrifuge tube, 50-200 μ L is vacantly added dropwise to the middle part of adsorbed film
Elution buffer TE, is placed at room temperature for 2-5min, and solution is collected into centrifuge tube, -20 DEG C of preservations by 12000rpm centrifugation 2min.
PCR and sequencing
(1) DNA of extraction is with reference to the 16s rDNABacterial Identification PCR Kit of TaKaRa companies
Kit specification carries out PCR amplification, and wherein forward primer is：5’-AGAGTTTGATCATGGCTCAG-3’(SEQ ID
No.1), reverse primer is：3’-CGCTTACCTTGTTACGACTT-5’(SEQ ID No.2).
PCR amplification condition is as follows：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 1min；53 DEG C of annealing 1min；72 DEG C of extension 90s；
Extend 5min after 72 DEG C, totally 30 Xun Huans.
PCR product is separated using 1% agarose gel electrophoresis, and EB dyeing is placed on 3UVTMTransilluminator
It is observed under (UVP, USA).Find that LX13 bacterial strain 16s rDNA clip sizes are about 1500bp by electrophoresis detection, segment is big
Small to be consistent with kit desired design, amplification is shown in Fig. 2.
(2) according to the primer being pre-designed and expected amplified fragments size, binding marker (Marker) target fragment is in purple
It is cut down under outer lamp, the recycling of DNA is carried out with QIAquick Gel Extraction Kit Silica Bead DNAGelExtraction Kit.16s
RDNA sequencings carry out sequencing by precious bioengineering (Dalian) Co., Ltd.It measures sequence results and shows LX13 bacterial strains
16srDNA segments share 1456nt base-pairs composition, and sequence table information is shown in SEQ ID No.3.By blast program by measure
Sequence and GenBank (NCBI, website http://blast.ncbi.nlm.nih.gov/Blast.cgi) in sequence compare,
Then obtain from GenBank and be determined with test strain Sequences similar kind, the 16s rDNA sequences belonged to.Comparison result is shown in figure
3, the bacillus subtilis of bacillus in the 16s rDNA sequence GenBank gene pools of the results show active bacterial strain
(Bacillussubtilis) 16s rDNA sequence very high homologies, homology is up to 99%.Structure development tree and homology analysis
Bacillus subtilis (Bacillus subtilis) (Accession of the results show (Fig. 3), bacterial strain LX13 and bacillus
No:EF47226.1) it is separately formed a branch, closest in evolution, affiliation is nearest between reflecting them, fortune
It is 99% to analyze to obtain the two homology with DANMAN softwares.With reference to traditional physio-biochemical characteristics identification and 16S rDNA sequences
Arrange analyzing as a result, judging that bacterial strain LX13 is bacillus subtilis.
The preparation of 2 compound biocontrol fungicide of embodiment
(1) bacterial strain activates：The above-mentioned original microbial strains for being stored in inclined-plane are aseptically inoculated in pair respectively
It is activated on the culture medium answered.
Brevibacillus laterosporus-LX12, bacillus subtilis-LX13 are inoculated into 28 DEG C of culture 3d of LB solid mediums.
(2) antagonistic effect of biocontrol microorganisms：Using tablet face-off method, by Brevibacillus laterosporus-LX12 and bacillus subtilis
Bacterium-LX13 carries out antagonistic effect.It does not find to generate antagonism.
(3) seed culture：The training of LB liquid is accessed after Brevibacillus laterosporus-LX12, bacillus subtilis-LX13 are activated
Base is supported, is placed in 150r/min shake cultures 3d in 28 DEG C of shaking tables.
(4) fermenting and producing of microbial inoculum：Beef extract-peptone fluid nutrient medium is taken, is respectively connected to the side of above-mentioned preparation
Spore bacillus brevis-LX12, bacillus subtilis-LX13 fermentation seed liquids, in 30~40 DEG C of pH7.2~8.5, temperature, fermentation 1
~2d is to get spare as production bacterium solution.
(5) preparation of liquid compound biocontrol fungicide：Production bacterium solution prepared by above-mentioned steps, according to the short gemma bar of side spore
The volume ratio of bacterium-LX12 zymocyte liquids and bacillus subtilis-LX13 zymocyte liquids is that the proportioning of 2 ︰ 1 is mixed, and is made multiple
Close biocontrol agent.
Through examining, the total living bacteria count of compound biocontrol fungicide of preparation is up to 200,000,000/milliliter, the quantity of each effective bacterium
20,000,000/milliliter or more must not be less than, meet standard GB/T -20287-2006.
3 indoor pot application compound biocontrol fungicide cure plant disease of peanut of embodiment and growth-promoting functions experiment
(1) activation of pathogen：The peanut crown rot bacterium of preservation, sclerotium rolfsii, pine root fungus are seeded to PDA culture medium
In, it cultivates 5 days, is activated in 28 DEG C of greenhouses.
The expansion of pathogen is numerous：Oat grain is fitted into triangular flask, moisture is outwelled after distilled water immersion 6h, 121 DEG C of high pressures are gone out
Bacterium 20min；Peanut crown rot bacterium, sclerotium rolfsii, pine root fungus are inoculated into the oat grain of sterilizing respectively, are placed in 28 DEG C of cultures
7d, is shaken every day inoculation bottle 2 times, makes the equal pathogen of all oat grains, obtains the oat grain that carries disease germs.
(2) pathogen is inoculated with：By peanut (spending No. 8 in kind Shandong) plantation in the basin of phjytotron, soil：Vermiculite：Battalion
Support mass ratio=2 of soil：1：1.Cultivation temperature daytime is 28 DEG C, and at 25 DEG C of night, 12h illumination/12h is dark, the plantation of peanut simple grain,
Per 3, basin.10 utilizations of wheat of carrying disease germs of each pathogen spread table local method and are inoculated in peanut periphery, and subsequent lid last layer thin soil simultaneously pours
Water.
(3) inoculation of compound biocontrol fungicide：Respectively the peanut seeding phase, after planting 10d using compound biocontrol fungicide, side spore
Bacillus brevis-LX12 zymotic fluids, bacillus subtilis-LX13 fermentation liquid irrigating roots, it is each to handle 10 basins, per 3 plants of basin, repeat 3
It is secondary.Pour the zymotic fluid 100m L of 100 times of dilution every time per basin.Two controls of 50% carbendazim, 800 times of liquid and clear water are set.Daily
Peanut incidence is observed, the incidence of crown rot, southern blight and root rot is investigated in plantation after 75 days.Peanut watering 1 daily
It is secondary.
5 grades of grade scales of crown rot：0 grade：Plant is asymptomatic；1 grade：Only scab is generated in basal part of stem；2 grades：Basal part of stem produces
Raw contracting symptom of hanging, less than 1/3rd performance systemic symptoms (withered, dead, wilting etc.) of whole strain；3 grades：The three of whole strain/
Less than two show systemic symptom；4 grades：More than 2/3rds representation system symptoms of whole strain, dead plant is by 4 grades of calculating.
5 grades of grade scales of southern blight：0 grade：Plant is asymptomatic；1 grade：Scab is only generated in basal part of stem；2 grades：Basal part of stem produces
Raw contracting symptom of hanging, the affected part of representation system symptom (withered, dead, wilting etc.) account for less than 1/3rd of whole strain；3 grades：
The affected part of representation system symptom accounts for less than 2/3rds of whole strain；4 grades：The affected part of representation system symptom accounts for whole strain
More than 2/3rds, dead plant is by 4 grades of calculating.
10 grade scale of root rot：0 grade:Equal disease-free spot on stem foot and main fibrous root；1 grade:There is a small amount of disease on stem foot and main root
Spot；3 grades:Scab is more on stem foot and main root, and lesion area accounts for the 1/4~1/2 of stem foot and the root gross area；5 grades:Stem foot and main root
Upper scab is more and big, and lesion area accounts for the 1/2~3/4 of stem foot and the root gross area；7 grades:Scab in flakes, is formed on stem foot and main root
Around stem phenomenon, but root system is not dead；9 grades:Root system necrosis, plant above ground portion wilts or death.
Diseased plant rate=morbidity strain number/total strain number × 100%
Disease index=∑ (morbidity grade typical value × diseased plant number at different levels) × 100/ (investigation total strain number × superlative degree morbidity generation
Tabular value)
Control effect=[(control disease index-processing disease index)/control disease index] × 100%
(4) experimental result：
1. the effect of cure plant disease of peanut
With the fermentation liquor treatment peanut bacterial strain of compound biocontrol fungicide, compound biocontrol fungicide is to indoor pot peanut crown rot
Control effect is significantly higher than the seed dressing of comparison medicament carbendazim, is also significantly greater than Brevibacillus laterosporus-LX12 fermentations up to 76.8%
Bacterium solution, bacillus subtilis-LX13 zymocyte liquids single dose apply (table 2)；Compound biocontrol fungicide is to indoor pot peanut sclerotium rolfsii
Control effect for 61.0%, preventive effect is higher than the seed dressing of comparison medicament carbendazim, Brevibacillus laterosporus-LX12 zymocyte liquids and withered
Careless bacillus-LX13 zymocyte liquids single dose applies (table 3)；Compound biocontrol fungicide imitates the prevention of indoor pot peanut root rot
Fruit is significantly higher than the seed dressing of comparison medicament carbendazim up to 70.1%, be also significantly greater than Brevibacillus laterosporus-LX12 zymocyte liquids,
Bacillus subtilis-LX13 zymocyte liquids single dose applies (table 4).This illustrates compound biocontrol fungicide to peanut crown rot, southern blight
There is preferable control effect with root rot, it is especially prominent to the control effect of peanut crown rot and root rot, more than 70%.
2 compound biocontrol fungicide of table is to the control effect (potting) of peanut crown rot
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
3 compound biocontrol fungicide of table is to the control effect (potting) of peanut sclerotium rolfsii
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
4 compound biocontrol fungicide of table is to the control effect (potting) of peanut root rot
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
2. promote the effect of Development of Peanut
Respectively sowing time, after planting 10d using compound biocontrol fungicide zymotic fluid, Brevibacillus laterosporus-LX12 ferment
Liquid, bacillus subtilis-LX13 fermentation liquid irrigating roots, it is each to handle 10 basins, per 3 plants of basin, it is repeated 3 times.Dilution 100 is poured every time per basin
Times zymotic fluid 100ml, to pour 800 times of liquid of same volume clear water and 50% carbendazim as control.Peanut seeding is received after 4 months
It obtains, pours out potting peanut, measurement peanut stem height, side shoot length, ground diameter, branch amount；Peanut pod number is recorded, is washed with flowing water
It naturally dry and weighs after pod surface soil particle.Peanut plant ground, under ground portion are cut off with scissors, is put into drying box
Its dry weight is weighed after drying to constant weight at 80 DEG C.
It the results are shown in Table 5：The peanut of inoculation compound biocontrol fungicide is averaged stem height, side shoot length, single plant pod number, pod weight
Amount, overground part dry weight, underground part dry weight are obviously higher than blank peanut, and on ground diameter is thick, branch amount does not influence；Single plant pod
Number, pod weight is substantially better than Brevibacillus laterosporus-LX12 zymotic fluids, bacillus subtilis-LX13 zymotic fluids are individually handled
Peanut, illustrate that compound biocontrol fungicide significantly facilitated the growth and development of peanut, and improve the yield and quality of peanut.
5 compound biocontrol fungicide Y-1 of table is inoculated with the influence (potting) to Development of Peanut
Compound biocontrol fungicide cure plant disease of peanut is applied in 4 field of embodiment and growth-promoting functions are tested
Experiment is carried out in Shandong Peanut Inst. Laixi experimental plot, for peanut continuous cropping field, peanut crown rot, southern blight
It is serious with the generations such as root rot.Using RANDOMIZED BLOCK DESIGN, peanut is per cell 70m2, it is repeated 4 times.Clear water is blank control,
50% 800 times of carbendazim liquid is positive control.Respectively sowing time, after planting 10d using compound biocontrol fungicide ferment liquid irrigating root,
Pour 100 times of zymotic fluid about 100ml of dilution every time per pier.Other field management are produced with normal, are investigated before harvesting peanut per cell
Middle row looks into 40 plants per cell, looks into 160 plants altogether, the incidence of investigation peanut crown rot, southern blight and root rot；Per small
Receive 12m in area2, investigate peanut yield.
Control effect result of the test：The fermentation liquor treatment peanut of field compound biocontrol fungicide prevents peanut crown rot
Effect is 66.6%, is significantly higher than the seed dressing of comparison medicament carbendazim, is also significantly greater than Brevibacillus laterosporus-LX12 zymocyte liquids
(table 6) is applied with bacillus subtilis-LX13 zymocyte liquids single dose；Control effect to peanut sclerotium rolfsii is 56.7%, significantly
Dress seed higher than comparison medicament carbendazim, be also significantly greater than Brevibacillus laterosporus-LX12 zymocyte liquids and bacillus subtilis-
LX13 zymocyte liquids single dose applies (table 7)；It is 63.6% to peanut root rot control effect, is significantly higher than comparison medicament carbendazim
Seed dressing is also significantly greater than Brevibacillus laterosporus-LX12 zymocyte liquids, bacillus subtilis-LX13 zymocyte liquid single dose applications
(table 8).Compound biocontrol fungicide is significantly higher than comparison medicament carbendazim to peanut crown rot, southern blight and root rot control effect,
Also significantly greater than Brevibacillus laterosporus-LX12 zymocyte liquids and bacillus subtilis-LX13 zymocyte liquid single dose applications, explanation
Brevibacillus laterosporus-LX12 and bacillus subtilis-LX13 have been used in mixed way synergistic function.
6 compound biocontrol fungicide of table is to the field control effect of peanut crown rot
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
7 compound biocontrol fungicide of table is to the field control effect of peanut sclerotium rolfsii
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
8 compound biocontrol fungicide of table is to the field control effect of peanut root rot
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
Yield trials the results are shown in Table 9：The peanut yield for being inoculated with compound biocontrol fungicide increases production 49.22% compared with blank control, compared with
Using the processing volume increase 15.13% of carbendazim seed dressing.Illustrate that using compound biocontrol fungicide peanut yield can be significantly increased.Using
The yield of compound biocontrol fungicide be also significantly greater than Brevibacillus laterosporus-LX12 and bacillus subtilis-LX13 single doses application
Yield.
9 compound biocontrol fungicide of table is inoculated with the influence to peanut yield
The preparation of 5 solid union biocontrol agent of embodiment
Charcoal is prepared by raw material of peanut vine, charcoal crushed 30 mesh sieves obtains biological powdered carbon, will obtain biology
Powdered carbon is 1 according to solid-to-liquid ratio:2 ratio is put into compound biocontrol fungicide, is stirred evenly, air drying, is obtained solid multiple
Close biocontrol agent.
Solid union biocontrol agent cure plant disease of peanut is applied in 6 field of embodiment and growth-promoting functions are tested
Experiment is carried out in Shandong Peanut Inst. Laixi experimental plot, for peanut continuous cropping field, peanut crown rot, southern blight
It is serious with the generations such as root rot.Using RANDOMIZED BLOCK DESIGN, peanut is per cell 70m2, it is repeated 4 times.Clear water is blank control,
50% 800 times of carbendazim liquid is positive control.The sowing of solid union biocontrol agent is carried out in sowing time, is sowed every time per pier
0.5 gram, topsoil is turned over, and be allowed to be sufficiently mixed with microbial inoculum, soil moisture content is kept between 15-25%.Other
Field management is produced with normal, investigates middle row before harvesting peanut per cell, and 40 plants are looked into per cell, looks into 160 plants altogether, investigation is spent
The incidence of raw crown rot, southern blight and root rot；12m is received per cell2, investigate peanut yield.
Control effect result of the test：Solid union biocontrol agent is 65.0% to peanut crown rot control effect, significantly high
It dresses seed in comparison medicament carbendazim, is also significantly greater than the compound biocontrol fungicide (table 10) of liquid；The prevention of peanut sclerotium rolfsii is imitated
Fruit is 54.5%, is significantly higher than the seed dressing of comparison medicament carbendazim, is also significantly greater than the compound biocontrol fungicide (table 11) of liquid；To flower
Maize ear rot of taking root control effect is 61.9%, is significantly higher than the seed dressing of comparison medicament carbendazim, also significantly greater than liquid
Compound biocontrol fungicide (table 12).Solid union biocontrol agent is long compared with the liquid compound biocontrol fungicide term of validity, can be longer by one
It persistently plays a role in the section time, the entire peanut growth phase only needs to apply once, saves production cost.
10 solid union biocontrol agent of table is to the field control effect of peanut crown rot
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
11 solid union biocontrol agent of table is to the field control effect of peanut sclerotium rolfsii
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
12 solid union biocontrol agent of table is to the field control effect of peanut root rot
Note:In table in same row between different lowercase letters processing difference up to 5% level of signifiance
Yield trials the results are shown in Table 13：The peanut yield for being inoculated with solid union biocontrol agent increases production compared with blank control
53.55%, relatively using the processing volume increase 18.47% of carbendazim seed dressing, compared with the volume increase for applying liquid compound biocontrol fungicide
13.08%.Illustrate that using solid union biocontrol agent peanut yield can be significantly increased.
13 solid union biocontrol agent of table is inoculated with the influence to peanut yield
Sequence table
<110>Shandong Peanut Inst.
<120>A kind of preparation method and application of the compound biocontrol fungicide of cure plant disease of peanut
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
agagtttgat catggctcag 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cgcttacctt gttacgactt 20
<210> 3
<211> 1454
<212> DNA
<213>Bacillus subtilis (Bacillus subtilis)
<400> 3
caggacgaac gcgcggcgtg cctaatacat gcaagtcgag cggacagatg ggagcttgct 60
ccctgatgtt agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga 120
taactccggg aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcaaacata 180
aaaggtggct tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg 240
taacggctca ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg 300
actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg 360
aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt 420
gttagggaag aacaagtacc gttcgaatag ggcggtacct tgacggtacc taaccagaaa 480
gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 540
attattgggc gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct 600
caaccgggga gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc 660
cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc 720
tggtctgtaa ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg 780
gtagtccacg ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg 840
cagctaacgc attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga 900
attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 960
ccttaccagg tcttgacatc ctctgacaat cctaggagat aggacgtccc ctttcggggg 1020
cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080
ccgcaacgag cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga 1140
ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac 1200
ctgggctaca cacgtgctac aatggacaga acaaagggca gcgaaaccgc gaggttaagc 1260
caatcccaca aatctgttct cagttcggat cgcagtctgc aactcgactg cgtgaagctg 1320
gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac 1380
accgcccgtc acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttttagga 1440
gccagccgcc gaag 1454
Claims (6)
1. a kind of preparation method of the compound biocontrol fungicide of cure plant disease of peanut, which is characterized in that the compound biocontrol fungicide
Strain component be Brevibacillus laterosporus-LX12, deposit number be CGMCC 7420 and bacillus subtilis-LX13, preservation
Number is CGMCC No.8805；Its preparation method is trained will to be inoculated into 28 DEG C of LB solid mediums after above-mentioned actication of culture respectively
Support 3d；Beef extract-peptone fluid nutrient medium is taken, is respectively connected to the Brevibacillus laterosporus-LX12 fermentation seed liquids of above-mentioned preparation
With bacillus subtilis-LX13 fermentation seed liquids, in 30~40 DEG C of pH7.2~8.5, temperature, 1~2d of fermentation uses to get production
Bacterium solution；Various production bacterium solutions prepared by above-mentioned steps, according to Brevibacillus laterosporus-LX12 bacterium solutions and bacillus subtilis
The volume ratio of bacterium-LX13 bacterium solutions is 2:1 proportioning mixing, is made compound biocontrol fungicide.
2. preparation method according to claim 1, which is characterized in that charcoal is prepared by raw material of peanut vine, by biology
Charcoal crushed 30 mesh sieves and obtain biological powdered carbon, and will obtain biological powdered carbon according to solid-to-liquid ratio is 1:The ratio of (1~3) is put into compound
It in biocontrol agent, stirs evenly, air drying, obtains solid compound biocontrol fungicide.
3. preparation method according to claim 1, which is characterized in that charcoal is prepared by raw material of peanut vine, by biology
Charcoal crushed 30 mesh sieves and obtain biological powdered carbon, and will obtain biological powdered carbon according to solid-to-liquid ratio is 1:The ratio of (1~3) is put into respectively
In Brevibacillus laterosporus-LX12 bacterium solutions and bacillus subtilis-LX13 bacterium solutions, the Brevibacillus laterosporus-LX12 bacterium solutions
Volume ratio with bacillus subtilis-LX13 bacterium solutions is 2:1, it stirs evenly, air drying, obtains solid biological and ecological methods to prevent plant disease, pests, and erosion single dose, it will
Two kinds of single doses are mixed to get solid compound biocontrol fungicide.
4. claims 1 to 3 any one of them method prepare compound biocontrol fungicide prevention peanut crown rot, southern blight and
Application in terms of root rot and the application in terms of peanut growth and output increased is promoted.
5. application according to claim 4, which is characterized in that respectively the sowing time in peanut, after planting 10d is using compound
Pouring root or the sowing time in peanut carry out the sowing of solid union biocontrol agent after biocontrol agent dilution.
6. application according to claim 5, which is characterized in that each per pier peanut by 100 times of compound biocontrol bacterium dilution agent
Pour 100ml.
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| CN115074283A (en) * | 2022-06-29 | 2022-09-20 | 河南科技大学 | Brevibacillus laterosporus and application thereof |
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